Mycobacterium kansasii (Mk) is among the six most common nontuberculous mycobacterial pathogens associated with opportunistic pulmonary infections in the United States and many other areas of the world, where it is often surpassed only by the Mycobacterium avium complex. Mk causes chronic pulmonary disease with tuberculosis (TB)-like features and mortality associated with treatment failure and comorbidities. The prevalence of Mk chronic pulmonary disease remains underrepresented, particularly in high TB-human immunodeficiency virus (HIV) burden areas. Yet it is clear that Mk infections continue to rise due to the uncurbed HIV epidemic and the global increase of other predisposing factors in populations around the world. Worrisomely, the juxtaposition of the HIV and TB epidemics creates fertile grounds for the rise of drug resistant Mk strains, and Mk isolates resistant to anti-TB drugs that are also a cornerstone of anti-Mk treatments have been reported. Drug resistance is a threat to the future control of Mk infections, which already requires long- term, multidrug courses with adverse side effects and challenging compliance. This bleak scenario makes development of new and more efficacious anti-Mk therapeutics of utmost importance. To this end, a comprehensive understanding of the unexplored Mk biology is critical to better position the community to address the current public health challenges arising from Mk infections and the inevitable upcoming of multidrug resistant Mk strains as a collateral outcome of the pervasive use of anti-TB drugs to curb the TB epidemic. In particular, elucidation of the essential genes in Mk will help illuminate and prioritize new potential drug target candidates and avenues to the development of novel therapeutics against Mk. With these considerations in mind, a long-term goal set for our Mk research is to apply genetic approaches to interrogate Mk gene essentiality. The underlying central hypothesis behind this R03 project is that methodologies combining transposon (Tn) mutagenesis with next-generation sequencing-based insertion site identification (hereafter referred to as ?Tn Insertion Sequencing?, TIS) are powerful tools to unravel gene essentiality in Mk. To begin testing this hypothesis, we propose the generation and analyses of Mk genome-wide scale TIS- derived data to predict genes essential for growth in rich laboratory medium. Encouragingly, our preliminary data establish proof-of-principle and technical feasibility for this project. Upon completion, this project will result in the development of a TIS approach for Mk and deliver new knowledge to the field of Mk gene essentiality. The availability of this knowledge to the research community will support new potential drug target candidate identification/prioritization endeavors, benefit current Mk genome annotation efforts, expand comparative functional genomic capabilities and existing essential gene databases, and aid Mk computational systems biology pipelines for metabolic network reconstructions and physiology modeling. Thus, the outcomes of this project will advance the field and benefit the mycobacterial research community and beyond.